Defining regenerative potential in the cardiac conduction system PROJECT SUMMARY Many cardiac arrhythmias result from damage to the cardiac conduction system (CCS), which orchestrates car- diac electrical activity to ensure regular contractile function. Similar to atrial and ventricular myocytes, CCS cells arise from cardiomyocyte progenitors, but in contrast they undergo terminal differentiation and cell-cycle exit prior to birth. Although regenerative capacity is strongly correlated with active cell division, the regenerative potential of the CCS has not been directly evaluated. The long-term goal of our research program is to devise new therapeutic approaches for acquired arrhythmias. The overall objective of this proposal is to examine the neonatal regenerative capacity of the atrioventricular conduction system (AVCS), a CCS structure that coordi- nates atrioventricular (AV) synchrony. There is an urgent need to elucidate the cellular and molecular underpin- nings of AVCS regeneration to establish a potentially new pathway for treatment of cardiac arrhythmias. Using a novel genetic system for ablating AVCS cardiomyocytes generated in our lab, we found that adult AVCS abla- tion results in persistent atrioventricular (AV) conduction defects, contractile dysfunction, and a failure to regen- erate. In contrast, neonatal AVCS injury led to spontaneous recovery from subtotal injury, providing the first definitive evidence for regenerative potential within the CCS. Building on this observation, our central hypothesis is that the AVCS regenerates by reconstructing its native configuration via proliferation and electrical remodeling of pre-existing cardiomyocytes. To test our hypothesis, we propose the following Specific Aims: 1) Determine the cellular mechanisms that underlie AVCS regeneration, 2) Define the functional role of Gata4/6 during AVCS regeneration, and 3) Establish molecular mechanisms by which Gata4/6 regulate AVCS regeneration. In Aim 1, we will use our AVCS injury system and cell type-specific immunostaining markers to identify the major cell types that contribute to AVCS regeneration. In Aim 2, we will use our AVCS injury system in conjunction with Gata4/6 floxed and conditional overexpression alleles to characterize their role during AVCS regeneration. In Aim 3, we will perform immunostaining, cellular electrophysiology, and RNA-Seq analysis to define the molecular mecha- nisms by which Gata4/6 influence AVCS regeneration. Successful completion of the proposed project will de- lineate critical cellular and molecular features of AVCS regeneration. This contribution will be significant because such insight will establish proof-of-concept that regeneration can impact recovery from dysrhythmia. Further- more, the proposed research is innovative because our unique set of transgenic tools enables detailed in vivo interrogation of AVCS regeneration to establish a potentially new therapeutic paradigm for resolution of cardiac dysrhythmia. Taken together, we anticipate that the results of the proposed project will define critical cellular and molecular features of AVCS regeneration and establish a foundation for future pathway-specific studies and anti- arrhythmic drug development.